The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness. T. brucei has served as a model system for the study of the related pathogens Trypanosoma cruzi and Leishmania spp. Due to the breakdown of vector control, African sleeping sickness is dramatically increasing in incidence. There is no vaccine for this disease, which is invariably fatal if untreated. The emergence of drug resistant strains increases the need for new therapeutic options. The project focuses on phosphoproteins of trypanosomes, and their role in critical cellular functions such as proliferation and development. Such functions could serve as important targets for drug discovery. Precedents in other organisms demonstrate the necessity of identifying a batter of signaling molecules and their targets as a prelude to efficiently defining the regulatory circuits controlling cell function. Our initial studies examined a phosphoprotein, Nopp44/46, and a protein kinase, NrkA. NrkA is developmentally regulated and that it contains a PH domain. Nopp44/46 is a nucleolar phosphoprotein that is developmentally regulated at the level of protein abundance and tyrosine phosphorylation. The identification of candidate molecules with which these proteins interact, including a novel G protein and casein kinase 2, provides impetus for expansion of these studies. The focus of the next grant period will be to further examine the function of each of these molecules using genetic approaches. Within the context of our specific aims, will be development new approaches to examine protein interactions in vivo, to regulate gene expression, and to explore parasite functions using yeast as a cellular test tube. Interwoven are studies of nucleolar function and proliferation. This will form a scaffold to which new findings can be linked, and allow phosphorylation pathways in T. brucei to be elucidated.